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Abstract

The electroretinogram (ERG) is a sensitive and noninvasive method for testing retinal function. In this protocol, we describe a method for performing ERGs in mice. Contact lenses on the mouse cornea measure the electrical response to a light stimulus of photoreceptors and downstream retinal cells, and the collected data are analyzed to evaluate retinal function.

Electroretinograms (ERGs) are used by researchers and clinicians to test retinal function by measuring the electrical response of retinal cells to a light stimulus. The ERG is a useful tool for measuring retinal responses in mice due to its high level of sensitivity and noninvasive nature, and can be utilized to assess eye disease and retinal degeneration (Duncan et al., 2003; Phillips et al., 2010; Zhao et al., 2011; Vollrath et al., 2015). In mouse genetic retinal disease models, ERGs can be used to assess retinal degeneration at multiple time points as the disease progresses (Duncan et al., 2003). For studies evaluating the effect of drug treatment on the mouse eye, retinal function can be assessed before and after treatment in the same eye (Zhao et al., 2011). In the following protocol we describe a method for measuring the functional response of photoreceptors and downstream retinal cells in mice that builds on a previously published approach (Phillips et al., 2010). The protocol can be readily applied to animals three weeks of age and older.

ERGs can be used to measure the electrical response to light flashes in either dark-adapted (scotopic) or light-adapted (photopic) mice. In scotopic ERGs, mice are presented with low intensity light flashes to induce rod activation, so the function of rod photoreceptor and downstream retinal cells can be examined (Fu, 2010). A prolonged period of dark-adaptation is critical to achieve maximal rod sensitivity, and to keep cone stimulation minimal (Pepperberg, 2003). In photopic ERGs, mice are presented with high intensity light flashes after a period of light stimulation. Under these conditions, there is high cone activation and the rod response is suppressed. Therefore, photopic ERGs can be used to measure the function of cone photoreceptors and downstream retinal cells (Fu, 2010).

This method of performing ERGs allows for the calculation of amplitude and time-to-peak of two major waves, the a-wave and b-wave. The a-wave is a measure of the initial response of photoreceptors to a brief flash of light (Brown, 1968; Perlman, 2015). The b-wave is a measure of the response of downstream retinal neurons, including bipolar cells, to photoreceptor stimulation (Brown, 1968; Perlman, 2015). Loss of amplitude in either the a-wave or b-wave may be attributed to a number of retinal dystrophies (Creel, 2015), while ‘supernormal’ waves with increased amplitude have been attributed to cone dystrophies (Phillips et al., 2010).

Warm the non-electric heat pad in a 37 °C bath or incubator overnight.

Prepping for the experiment

Set up the dark room

Existing light sources can be used if covered with red filter to make them procedure-safe.

Cover the computer monitor and any miscellaneous lights with red filter or black electrical tape.

Cover the 37 °C heat pad with an absorbent pad. Position the elevated platform with heat pad on top so that the mouse can be easily prepared and moved into the dome.Note: The heat pad may need to be reheated to 37 °C if multiple animals are tested.

Move mice to experimental dark room if different from overnight adaption room. This can be done using either a blackout cloth or light-proof box. Mice that are not being tested should be kept in a dark environment.

Set stimulus settings as described below.

Preparing the miceNote: From this point on, only red-filtered light sources should be used in order to keep mice dark-adapted.

Anesthetize the mouse by injection of ketamine hydrochloride/xylazine hydrochloride (80 mg/kg/13 mg/kg), and allow mice to become fully sedated (2-5 min). Check for a toe-pinch response to ensure the animal is adequately anesthetized.

Treat both eyes with 1% atropine sulfate, 2.5% phenylephrine hydrochloride, and 0.5% proparacaine hydrochloride, allowing drops to sit on the eyes for ~2 min before wicking with a cotton swab and applying the next drop. After this point the eyes should be kept hydrated with a hydrating eye ointment until the contact lens electrodes are applied (Figure 1).

Position the mouse on the heated platform. Place the ground needle electrode in the base of the tail, and reference needle electrode subdermally between the eyes (Figure 2).

Figure 2. Electrode placement on the mouse

Wick any excess moisture from the eyes, and add a small drop of 2.5% hypromellose to each eye.

Use fingers or forceps to position the contact lens electrodes onto the corneas. The hypromellose should keep the lenses in place. It may be helpful to tape down the electrode wires to the pad using surgical tape so they do not shift during the experiment (Figure 3, Video 1).

Figure 3. Contact lens electrodes on the mouse eye

Video 1. Applying the contact lens electrodes to the mouse eye

Slide the heated platform into the dome so that the mouse’s eyes are inside of the dome (Figures 4 and 5).

Figure 4. Mouse with electrodes on the heated, elevated platform

Figure 5. Mouse with eyes inside of the Ganzfeld ColorDome

Before beginning and throughout the scotopic and photopic assays, periodically check that impedance levels are acceptably low (within the green range). A low impedance level indicates that the electrodes have been properly installed. Note: The impedance may be high if any of the electrodes have shifted and the circuit has been broken. The Espion visual electrophysiology system displays impedance using green, yellow and red bars to signify the level of resistance in the circuit.

Run through each step of light intensity using the Espion E2 system.Note: Check the impedance every 2-3 steps or as needed to ensure that the electrode circuit has not been broken.

Running the photopic (light-adapted) ERG

Light adapt the mouse for at least 10 min at a light intensity of 20 cd sec/m2. Light adaptation saturates the rod response so that the cones, which account for only 3% of the photoreceptor population in the mouse eye, can be stimulated and recorded without rod interference (Fu, 2010). Note: It may be necessary to give the mouse a second, reduced dose of ketamine/xylazine if the animal begins to wake up.

Remove electrodes from the mouse and gently clean contact electrodes with a cotton swab and water. Needle electrodes can be wiped with 70% ethanol.

Place mouse in a clean cage on top of a heat pad until it recovers. Keep the mouse’s eyes moist with a hydrating eye ointment (Figure 6).

Figure 6. Mouse recovering in a clean, heated cage

Data analysis

Processing ERG data (Figures 7 and 8)

Data from two eyes can either be averaged for a single animal, or analyzed separately if one eye is experimental and one is control.

Use a data analysis software such as Microsoft Excel or GraphPad Prism to generate traces.

a-wave:

The a-wave amplitude can be measured by calculating the difference in trace amplitude between time (t) = 0 and the lowest point on the trace.Note: The tail of the trace may have points lower than the bottom of the a-wave, which is the first wave to be recorded. Exclude these tail points if this is the case.

The a-wave latency of response (time-to-peak) can be measured by calculating elapsed time between t = 0 and the time point at the lowest point on the trace.

b-wave:

The b-wave amplitude can be measured by calculating the difference in trace amplitude between the bottom of the a-wave and the top of the tallest curve.Note: There may be oscillating potentials that rise above the top of the b-wave curve. Exclude these points, as they will give an inaccurate b-wave if included. Oscillating potentials are thought to reflect cell activity in the inner retina, and can be isolated using a digital filter (Asi and Perlman, 1992).

The b-wave implicit time (time-to-peak) can be measured by calculating elapsed time between the bottom of the a-wave and the top of the b-wave.

Figure 7. Representative ERG traces of C57BL/6J adult mice. A pulse intensity of 1 cd sec/m2 was used for measurement of the scotopic ERG, and a pulse intensity of 5 cd sec/m2 was used for measurement of the photopic ERG.

Figure 8. Defining the a-wave and b-wave on an ERG trace. The a-wave is measured by calculating the difference in trace amplitude between time (t) = 0 and the lowest point on the trace. The b-wave amplitude is measured by calculating the difference in trace amplitude between the bottom of the a-wave and the top of the tallest curve.

Statistical analysis of ERG data

a-wave and b-wave amplitude:Note: Statistical analysis of a-wave and b-wave amplitude shown is based on data in Vollrath et al. (2015), and may not be appropriate for every data set.

Compile calculated a-wave amplitudes at a single light intensity for scotopic ERGs in each experimental group and perform a two-way ANOVA with Bonferroni’s correction for multiple comparisons (Figure 9) (Vollrath et al., 2015).

Repeat the statistical analysis for each light intensity.

The following notation can be used to show p-value significance: ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05 (Figure 9). Note: We recommend using at least 5 animals per group to achieve statistical significance, although more may be necessary.

A significant difference in a-wave amplitude at one or more light intensities may indicate a difference in photoreceptor function between the two experimental groups. A significant difference in b-wave amplitudes may indicate a difference in the function of retinal cells downstream of the photoreceptors, including bipolar cells (Vollrath et al., 2015).

a-wave and b-wave time-to-peak:

Compile calculated time-to-peak at a single light intensity for each experimental group and perform a two-way ANOVA with Bonferroni’s correction for multiple comparisons.

Repeat the statistical significance at each light intensity for a-wave and b-wave time-to-peak for both scotopic and photopic ERGs.

A significant difference in a-wave or b-wave time at one or more light intensities may indicate a delay in photoreceptor response or downstream retinal signaling (Perlman, 2015).

Supported by grants from the Foundation Fighting Blindness, the Macular Degeneration Research Program of the BrightFocus Foundation, the Thome Memorial Foundation, and NIH grants R01 EY025790 and T32 EY20485.
This protocol should be cited as Benchorin G., Calton M.A., Beaulieu M.O., Vollrath D. Assessment of murine retinal function by electroretinography. Bio Protoc. 2017.

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